Purification and properties of alcohol oxidase from Tanacetum vulgare

Alcohol oxidase (alcohol: O₂ oxidoreductase) from leaves of Tanacetum vulgare has been purified 5150-fold to homogeneity on disc electrophoresis and gel electrofocussing. The enzyme which is probably flavoprotein, has molecular weight 180 000 daltons and is comprised of two sub-units of 94 000 and 75 000 daltons. It is active over a broad range (pH 5–9) and best accepts primary aliphatic alcohols with 6 to 10 carbons, especially those with a 2-ene group. K_m values for hex-trans-2-ene-1-ol, geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) and n-octanol were 0.19, 1.56 and 0.49 mM respectively. The significance of the enzyme in the formation of leaf aldehyde (hex-trans-2-ene-1-al) and in terpene metabolism is discussed.     

Studies on some aspects of peroxidase from submaxillary gland

A peroxidase has been purified 25- to 30-fold over crude homogenate from goat submaxillary gland, which shows a single band of protein on polyacrylamide gel electrophoresis at four different pH values (4.6–10.0). A molecular weight (M_r) of approximately 2 × 10⁴ per heme binding site has been found. The molecular weight of the enzyme determined by Sephadex-gel filtration method, appeared to be 4 × 10⁴. The sedimentation pattern of the purified enzyme shows a symmetrical peak, although there was evidence of some small heterogenous material near the meniscus. The sedimentation coefficient of the enzyme (s^o 20^wat 0.4% of the enzyme concentration) was found to be 4.18, which indicates the molecular weight of the enzyme to be approximately 6 × 10⁴.     

Purification and properties of acetate kinase from Clostridium thermoaceticum

Acetate kinase (ATP: acetate phosphotransferase EC 2.7.2.1) has been purified from Clostridium thermoaceticum. The enzyme of a specific activity of 282 μmoles min^-1 mg^-1 appeared homogeneous as judged from Sephadex chromatography and sedimentation velocity. Polyacrylamide gel electrophoretic patterns at pH 9.0 and 9.5 showed heterogeneity. Velocity curves obtained with varying amount of acetate were of the Michaelis-Menten type with an apparent K _m of 0.135 M. With varying amounts of ATP sigmoidal kinetic was observed (S_0.5=1.64 mM), suggesting cooperative binding of this substrate. The enzyme had only moderate thermal stability with a temperature optimum of about 60°C and exhibited a broken line in an Arrhenius graph. From gel filtration a molecular weight of about 60 000 daltons was estimated for the enzyme. The S_20w value was 6.0 S.     

Purification and properties of a heme-containing aldehyde dehydrogenase from Methylosinus trichosporium

Crude extracts of various methylotrophic bacteria contained a soluble phenazine methosulfate-linked aldehyde dehydrogenase. Procedures for the purification of an aldehyde dehydrogenase from extracts of the obligate methane-utilizing bacterium Methylosinus trichosporium are described. The purified enzyme is homogeneous as judged from polyacrylamide gel electrophoresis. The purified enzyme catalyzes the oxidation of straight-chain aldehydes (C₁-C₁₀ tested), aromatic aldehydes (benzaldehyde, salicylaldehyde), glyoxylate, and glyceraldehyde. Biological electron acceptors such as NAD⁺, NADP⁺, FAD, FMN, pyridoxal phosphate, and cytochrome c do not act as electron carriers. Sulfhydryl agents [p-chloromercuribenzoate, N-ethylmaleimide, 5,5-dithiobis (2-nitrobenzoic acid), and thioacetamide], cuprous chloride, cupric sulfate, and thiourea inhibited enzyme activity. The molecular weight of the enzyme as estimated by gel filtration is approximately 43,000 and as estimated by sedimentation equilibrium analysis, 50,000. The sedimentation constant (S₂₀, w) is 2.8. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 22,000. The purified enzyme is light brown and has an absorption peak at 410 nm. Reduction of the enzyme with sodium dithionite resulted in the appearance of peaks at 523 and 552 nm and a shift in the Soret peak from 410 to 412 nm was observed. These results suggest that the enzyme is a hemoprotein. There was no evidence that flavins were present as a prosthetic group. The amino acid composition of the enzyme is also presented. Antisera prepared against the purified enzyme are nonspecific; they cross-reacted with isofunctional enzyme from other methylotrophic bacteria on Ouchterlony double-diffusion plates.     

Purification and characterization of glyoxalase II from Hansenula mrakii

Glyoxalase II [S-(2-hydroxyacyl)glutathione hydrolase], one of the components of the glyoxalase system, catalyzes the hydrolysis of S-lactoylglutathione to glutathione and d-lactic acid. The enzyme was partially purified from the yeast Hansenula mrakii IFO 0895 by successive column chromatographies and polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 22,000 daltons by gel-filtration of Sephadex G-150 column chromatography and 24,000 daltons by SDS-polyacrylamide gel electrophoresis. The enzyme was specific to S-lactoyglutathione and S-acetylglutathione. The activity of the enzyme was strongly inhibited by Cu²⁺, p-chloromercuribenzoate and HgCl₂. The enzyme activity was also inhibited by hemimercaptal, a non-enzymatic condensation product between glutathione and methylglyoxal.     

Purification and characterization of beef liver dihydrofolate reductase.

Beef liver dihydrofolate reductase has been purified to homogeneity by using a methotrexate affinity column followed by gel filtration to remove several higher molecular weight proteins. Tightly bound dihydrofolate is removed by hydroxylapatite chromatography. The overall purification is 13,000-fold; the specific activity is 26 units·mg^?1, approximately 25 times higher than previously reported. The enzyme has been shown to be homogeneous by the following criteria: (i) discontinuous gel electrophoresis, (ii) sodium dodecyl sulfate-gel electrophoresis, (iii) velocity sedimentation, (iv) equilibrium sedimentation, and (v) methotrexate titration. The amino acid composition has been determined. Notable features include a single cysteine, three tryptophan and three histidine residues. The N-terminal amino acid is leucine. The molecular weight determined by equilibrium sedimentation is 22,500. The s_20,w⁰ is 2.08 × 10^?13 S and D_20,w⁰ = 10.93 cm²·s^?1. A frictional coefficient of 1.04 indicates that the enzyme is essentially spherical. An isoelectrical point of 6.80 was measured.     

Ribulose Diphosphate Carboxylase from Autotrophic Euglena gracilis

Ribulose 1,5-diphosphate carboxylase (RUDPcase) from autotrophically grown Euglena gracilis was purified to homogeneity as measured by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and immunoprecipitation reactions. The enzyme represented about 9% of total protein and 24% of soluble protein in the autotrophic cell. Light-grown, heterotrophic cells seemed to contain considerably less RUDPcase. Native carboxylase from autotrophic Euglena showed an s_{20, w} at low protein concentrations of 17 to 17.5, suggesting a molecular weight of >500,000 daltons. Upon denaturation, the enzyme dissociated into two subunits having different amino acid compositions and molecular weights of 59,000 and 12,000 daltons. Based upon the amino acid mass ratios, a quaternary organization of 7 to 8 large and 8 to 10 small subunits per native enzyme molecule was indicated.     

Size distribution of poly(A)-containing messencer RNA from free polysomes of HeLa cells

The sedimentation properties of pulse-labeled and long-term labeled mRNA from highly purified HeLa cell free-polysomes, selected for poly(A) content by two successive passages through poly(T)-cellulose columns, were analyzed under native and denatured conditions. The sedimentation profile of the mRNA on both sodium dodecyl SO₄-sucrose gradients and formaldehyde-sucrose gradients showed a broad distribution of components with estimated molecular weights ranging from 2 × 10⁵ to 5.5 × 10⁶ daltons and a weight-average molecular weight of 8.5 × 10⁵ daltons.     

Physicochemical features of hemoglobin of the crustacean, Triops longicaudatus

Hemoglobin from the notostracan, Triops longicaudatus, was purified and characterized physicochemically. Stoke's radius (77.7 Å) and the sedimentation coefficient (S_20,w = 17.1) were estimated by gel filtration chromatography and density gradient centrifugation, respectively and then used to calculate the molecular weight (600,400 daltons), the frictional ratio (1.33), and the diffusion coefficient (2.70). These physical parameters, along with data on the amino acid composition, are compared with similar data on other crustacean hemoglobins.     

Purification and properties of glucose isomerase of Actinoplanes missouriensis

Actinoplanes missouriensis produces an intracellular soluble glucose Isomerase. The soluble enzyme can be purified by a DEAE-cellulose beads columm with a onestep salt elution. The purified enyzme exhibited a molecular weight of approximately 80,000 daltons, being composed of two identical subunits of about 42,000 daltons each. The K_m for glucose is 1.33M, the K_m for frucotse is 1.67M. The enzyme has an optimal pH of 7.0. The presence of the cobalt ion is not required to produce optimal activity of the enzyme if the proper amount of magnesium is present.     

Biochemical Characterization of an Acetylcholine-hydrolyzing Enzyme from Bean Seedlings

An acetylcholine hydrolyzing enzyme was prepared and purified (40 times) from dwarf bean hypocotyl hooks. The purity of the enzyme was proved by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined to be 65,000 daltons. Enzyme activity was the highest at pH 8.0 and between 30 and 36 C. The enzyme had an apparent affinity constant (K_m) for acetylcholine of 460/micromolar. The affinity for substrate analogs increased from butyrylthiocholine to propionylthiocholine to acetylthiocholine. The enzyme activity was inhibited by choline, neostigmine, physostigmine, manganese, and calcium. Magnesium had no influence on the enzyme activity. We conclude that the enzyme from dwarf beans is an acetylcholinesterase (EC 3.1.1.7).     

Bovine adrenocortical self-phosphorylating protein kinase,SPK 380: Purification and characterization

Recently, we described the partial purification and characterization of a novel adrenocortical cyclic nucleotide-independent protein kinase, PK 380, that catalyzes the phosphorylation of an endogenous peptide (120,000 daltons) and a serine residue(s) of the α subunit (38,000 daltons) of the eucaryotic initiation factor eIF-2 (Y. Kuroda, W. C. Merrick, and R. K. Sharma, 1982, Arch. Biochem. Biophys.213, 271–275). In the present communication we describe the purification to apparent homogeneity and characterization of this protein kinase (SPK 380). As shown by sucrose density sedimentation, the native enzyme has a molecular weight of 356,000. The protein is composed of three identical subunits of M_r 120,000. Polyacrylamide-gel isoelectric focusing electrophoresis revealed a single peak with pI 4.5. SPK 380 self-phosphorylated a histidine residue(s) of its 120,000-dalton peptide. This reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mm Mn²⁺ or 10 mm Mg²⁺) were essential for optimum activity. Thiol reagents (N-ethylmaleimide, p-chloromercuriphenylsulfonic acid) inhibited the kinase, indicating a sulfhydryl-group requirement for enzyme activity.     

Effects of lipid removal on the molecular size and kinetic properties of bovine plasma arylesterase

A purified arylesterase preparation from bovine plasma was characterized to the extent that it has a partial specific volume of 0.91ml/g and an apparent z-average molecular weight of 440000. The relatively large magnitude of the former reflects the presence of phospholipids, cholesterol, triglycerides and β-carotene, the last-named being responsible for the pronounced yellow colour of the preparation. Removal of the lipid material is accompanied by a decrease in the apparent z-average molecular weight to 120000, the size of the smallest species detected by high-speed sedimentation equilibrium being in the vicinity of 70000 daltons: denaturation of the lipid-free preparation with 6m-guanidine hydrochloride caused essentially complete breakdown into subunits of this size. In kinetic studies on the enzyme the maximal velocity for the hydrolysis of phenyl acetate was found to increase by 60% on addition of 1 mm-Ca²⁺, with the K_m showing a concomitant decrease from 6.6 to 2.1 mm. Removal of lipid had no detectable effect on V_max. or K_m in either the presence or the absence of Ca²⁺. It is concluded that the bovine plasma arylesterase preparation is either a lipoprotein or an enzyme–lipoprotein complex with properties very similar to those of the α₁-lipoprotein or high-density lipoprotein (HDL₂) fraction of serum.     

Purification and subunit structure of mouse liver cystathionase

Cystathionase has been purified from mouse liver by ammonium sulfate precipitation, ethanol precipitation, column chromatography on DEAE-cellulose and on hydrox-ylapatite, as well as Sephadex G-200 gel filtration. These procedures yielded a chromatographically homogeneous enzyme which was purified more than 1000-fold relative to whole liver extract. Overall recovery was approximately 4%. The purified enzyme does not contain detectable carbohydrate and migrates as a single protein component on analytical disc gel electrophoresis. A sedimentation coefficient of 8.3 S has been determined for the active enzyme by rate zonal centrifugation in glycerol gradients. This value suggests a molecular weight for the native enzyme of approximately 160,000 g/mol, a value similar to that estimated by gel filtration. Following sodium dodecyl sulfate gel electrophoresis in the presence of reducing agent and at different gel concentrations, a single protein component with a molecular weight of 40,000 g/mol was obtained. Thus, the enzyme appears to consist of four subunits of equal size. The K_m value for cystathionine at pH 8.1, 37 °C, and in the presence of 1 mm dithioerythritol is approximately 1 mm.     

Diphosphopyridine nucleotide-linked D-lactate dehydrogenases from the horseshoe crab, Limulus polyphemus and the seaworm, Nereis virens. I. Physical and chemical properties

d-lactate dehydrogenase has been purified from horseshoe crab (Limulus polyphemus) skeletal muscle and the seaworm (Nereis virens). The purified Limulus dehydrogenase was shown to be a dimer, with a molecular weight of approximately 70 000. Sephadex gel filtration and equilibrium sedimentation yield molecular weights of about 80 000 and 70 000 respectively. Acid dissociation yields a molecular weight species of about 35 000. The native enzyme has an s^o₂₀^w of 3.95. Extrapolation of para-hydroxymercuribenzoate inhibition curves to 100% inhibition corresponds to two molecules of para-hydroxymercuribenzoate bound per molecule of enzyme. Studies on the stoichiometric binding of reduced coenzyme show two molecules bound per molecule of enzyme. The number of tryptic peptides has been found to be one-half that expected from the amino acid composition. The electrophoretic pattern of isoenzymic forms can be best interpreted as suggesting that the enzyme is dimeric. In vitro high salt, freeze-thaw hybridizations of the isolated Limulus muscle isoenzymes yield the electrophoretic pattern predicted by a dimeric structure.The physical properties ot Nereis lactate dehydrogenase have been found to be similar to those for the Limulus muscle lactate dehydrogenase.     

NADP-malic enzyme from maize leaf: purification and properties.

NADP-malic enzyme (EC 1.1.1.40), which is involved in the photosynthetic C⁴ pathway, was isolated from maize leaf and purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. At the final step, chromatography on Blue-Sepharose, the enzyme had been purified approximately 80-fold from the initial crude extract and its specific activity was 101 μmol malate decarboxylated/mg protein/min at pH 8.4. The enzyme protein had a sedimentation coefficient (s_20,w) of 9.7 and molecular weight of 2.27 × 10⁵ in sucrose density gradient centrifugation, and molecular weight of 2.26 × 10⁵ calculated from sedimentation equilibrium analysis. The molecular weight of the monomeric form was determined to be 6.3 × 10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the pyruvate carboxylation reaction, HCO₃^? proved to be the active molecular species involved. With all other substrates at saturating concentration, the following kinetic constants were obtained: K_m (malate), 0.4 mm; K_m (NADP), 17.6 μm; K_m (Mg²⁺), 0.11 mm. The maize leaf malic enzyme was absolutely specific for NADP. The Arrhenius plot obtained from enzyme activity measurements was linear in a temperature range of 13 to 48 °C, and the activation energy was calculated to be 9500 cal/mol.     

Purification and characterization of a type-I topoisomerase from cultured tobacco cells

Cultured tobacco (Nicotiana tabacum, var Xanthi) cells contain a topoisomerase that removes positive and negative supercoils from DNA. The enzyme has an estimated molecular mass of 30,000 daltons under denaturing conditions, but may exist as a multimeric protein in the native state. Activity is enhanced significantly by either MgCl₂ or CaCl₂, but other divalent cations are much less effective in stimulating DNA relaxation. The purified enzyme acts by altering the linking number in topological steps of one and is inhibited by berenil or camptothecin, not novobiocin. Taken together, these data identify this enzyme as a type I topoisomerase.     

Molecular and Enzymatic Properties of Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidase from Baker’s Yeast

Pyridoxamine (pyridoxine) 5′-phosphate oxidase purified from baker’s yeast was found to have a molecular weight of ca, 55,000 daltons based on polyacrylamide gel electrophoresis. The size of the enzyme subunit was analyzed by gel electrophoresis in the presence of sodium dodecylsulfate. This showed that the enzyme was composed of two nonidentical subunits with a molecular weight of 27,000 and 25,000 daltons. Fluorescence titration of the apoenzyme with FMN suggested that the holoenzyme contained one mol of FMN per mol of the enzyme. The K_m value of FMN for apoenzyme was calculated to be ca. 16 nm on both activities of pyridoxamine 5′-phosphate oxidase and pyridoxine 5′-phosphate oxidase.     

Pig liver fatty acid synthetase: purification and physicochemical properties.

This paper reports the first detailed study of the physicochemical properties of a fatty acid synthetase multienzyme complex from a mammalian liver. Fatty acid synthetase from pig liver was purified by a procedure including the following main steps: (i) preparation of a clarified supernatant solution (50,000 g), (ii) ammonium sulfate fractionation, (iii) DEAE-cellulose chromatography to separate 11 S catalase from the 13 S fatty acid synthetase, (iv) a preparative sucrose density gradient step to remove a 7 S impurity, and (v) a calcium phosphate gel step to remove an unusual yellow 16 S heme protein to yield a colorless preparation. The purified fatty acid synthetase was colorless and showed a single symmetrical peak in sucrose density gradient and conventional sedimentation velocity experiments. Fatty acid synthetase was very stable at 4 °C in the presence of 1 mm dithiothreitol and 25% sucrose. Extrapolation to zero protein concentration yielded values of S^o_20,w = 13.3 S and D^o_20,w = 2.60 × 10^?7cm²/s for the sedimentation and diffusion coefficients of the enzyme. Frictional coefficient values of 1.55 and 1.56 × 10^?7 cm, respectively, were calculated from the values for the sedimentation and diffusion coefficients. Based on these frictional coefficient values, the Stokes radius of the enzyme was calculated to be 82.4 Å. Sedimentation and diffusion coefficient data yielded a molecular weight value of $\text{M}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{s}\text{D}\text{) = 478,000}$ and sedimentation equilibrium data yielded a value of M_w = 476,000. Preliminary intrinsic viscosity measurements at 20 °C gave a value of 7.3 ml/g, indicating that the enzyme is somewhat asymmetric. This is supported by the value of 1.58 calculated for the frictional ratio and by the fact that the values for the sedimentation and diffusion coefficients are both slightly lower than expected for a globular protein of molecular weight 478,000. The enzyme possesses about 90 SH groups per molecule, assuming a molecular weight of 478,000. The ultraviolet absorption spectrum of the enzyme shows a maximum at 280 nm and an unusual shoulder at 290 nm. The fluorescence spectrum of the enzyme is dominated by tryptophan fluorescence and, over the excitation range of 260–300 nm, there is a single emission maximum at 344 nm.     

N 6 -( 2 -isopentenyl) adenosine: biosynthesis in vitro in transfer RNA by an enzyme purified from Escherichia coli

An enzyme has been partially purified from Escherichia coli which catalyzes in vitro the transfer of the Δ²-isopentenyl group from Δ²-isopentenyl pyrophosphate to an adenosine residue in Mycoplasma sp. (Kid) tRNA. The product of the reaction is N⁶-(Δ²-isopentenyl) adenosine, which is known to be absent in this Mycoplasma tRNA. The enzyme has an approximate molecular weight of 55,000 daltons, requires reduced sulfhydryl groups and a divalent metal ion for full activity, and is specific for tRNA.